In recent years, there has been a growing interest in being health conscious among people and thus oligosaccharides, which are sweet carbohydrates having physiological effects different from those of sugars, have been actively developed. Oligosaccharides generally have two or more and less than ten constituent sugars in a polymerized form. There are oligosaccharides that are digested by a digestive enzyme (digestible oligosaccharides) and oligosaccharides that are not digested or are not easily digested to a high degree (nondigestible oligosaccharides). Most oligosaccharides with added values to health that have been developed are not digested by a digestive enzyme or are nondigestible oligosaccharides that are not easily digested to a high degree.
Nondigestible oligosaccharides are used in health foods such as foods for specified health uses. These nondigestible oligosaccharides are metabolized through a pathway different from that of digestible carbohydrates such as sucrose and starch. A nondigestible oligosaccharide taken orally reaches the large intestine without being digested by α-amylase or a small intestinal mucosal disaccharidases. In the large intestine, the nondigestible oligosaccharide is fermented by indigenous intestinal bacteria and metabolized into short-chain fatty acids such as acetate, propionate, and n-butyrate, carbon dioxide, hydrogen gas, methane gas, bacterial cell components, and the like. Among them, short-chain fatty acids are absorbed from the large intestine and used solely as the energy source of the host. That is, even carbohydrates that are not digested and absorbed provide energy to a living body by being fermented and absorbed in the large intestine.
At present, 741 items have been approved as foods for specified health uses, and 93 items contain nondigestible oligosaccharides (as of Nov. 26, 2007). Nine kinds of nondigestible oligosaccharides are used in the foods for specified health uses as components involved. However, the determination thereof is performed by original methods of manufacturers, and a standard determination method has yet to be established. The reason for this is as follows. Nondigestible oligosaccharides contain not only oligosaccharides that are not easily digested in the small intestine to a high degree but also oligosaccharides that are partly digested. Therefore, since nondigestible oligosaccharides have constituent sugars and structures different from each other, there are differences in the stability of each of the oligosaccharides and the action mechanism of hydrolases.
An enzyme-gravimetric method of Prosky, which is a dietary fiber determination method, is a method in which digestible carbohydrates and proteins are completely digested by hydrolases and the weight of the undigested matter left and having a large molecular weight is measured. Dietary fiber, which is a high polymer, can be determined by an established method such as the enzyme-gravimetric method of Prosky, but nondigestible oligosaccharides and sugar alcohols, which are low-molecular-weight compounds, cannot be determined by the enzyme-gravimetric method. In recent years, nondigestible oligosaccharides and sugar alcohols have been used in various health foods. Thus, a determination method that can also determine such nondigestible oligosaccharides and sugar alcohols has been considered.
One of the methods for determining a nondigestible oligosaccharide that are approved in AOAC (Association of Official Analytical Chemists) is an enzymatic-HPLC method achieved by partly changing the dietary fiber determination method that uses the enzyme-gravimetric method of Prosky (refer to FIG. 6). As shown in FIG. 6, the enzymatic-HPLC method is a method in which, as in the dietary fiber determination method, digestible components are hydrolyzed by hydrolases and then nondigestible low-molecular-weight substances that are not precipitated with 78% of ethanol are analyzed and determined by HPLC using a specific column. In this method, because an enzyme treatment process in a gastrointestinal tract of a living body is assumed, the amount of nondigestible oligosaccharides that reach the large intestine without being digested in the small intestine, that is, the amount of nondigestible oligosaccharides that are utilized by intestinal bacteria can be estimated.
[Patent Document 1] Japanese Patent No. 3183500